The present invention provides peptide antagonists of xcex1-melanocyte stimulating hormone and methods of inhibiting the effects of xcex1-melanocyte stimulating hormone on cells or tissues sensitive to that hormone, including lightening skin, modulating the immune response and treating malignant melanoma.
Skin pigmentation and tanning are related to the amount and distribution of melanin in epidermal melanosomes. The epidermis cell population includes keratinocytes and the melanocytes that supply the keratinocytes with melanosomes, i.e., melanin containing pigment granules, via dendritic processes. Melanin is a dark pigment that is produced by the oxidation of tyrosine to dopa and dopaquinone by the enzyme tyrosinase, to produce compounds that polymerize to form melanin.
There are a number of localized hyperpigmentation disorders that presently lack any safe and effective method of cosmetic treatment. These include pigmented spots such as ephelides (freckles), solar lentigines (also called liver spots), acanthosis nigricans (a hypermelanotic disorder), cafe-au-lait spots, nevi (moles), and melasma (localized post-partum darkening of the skin). There is also a continuing interest and need for a method for regulating pigmentation tone for the total skin surface, either for cosmetic purposes, e.g., to lighten the complexion, or to block the deleterious effects on the appearance caused by certain endocrine disorders. Heretofore, no safe and effective method for achieving the regulation of epidermal pigmentation tone has been available.
The endogenous hormone that is implicated in the regulation of pigmentation is xcex1-melanocyte stimulating hormone (xe2x80x9cxcex1-MSHxe2x80x9d) (Lerner et al., 1961, Nature 189, 176-179). xcex1-MSH is also implicated in the regulation of central nervous system and immune system functions (De Weid, 1993, Ann. NY Acad. Sci. 680, 20-28; Tatro, 1990, Brain Res. 536, 124-132; Luger, et al.,1993, Ann. N.Y. Acad. Sci. 680, 567-570; Cannon, et al., 1986, J. Immunol. 137, 2232-2236; Murphy et 20 al., 1983, Science 221, 192-193), growth (Strand, F. l., et al., 1993, Ann. NY Acad. Sci 680, 29-49), mitogenesis (Halaban et al., 1993, J. Ann. NY Acad. Sci. 689, 290-300), and melanoma (Varga et al., 1974, Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593).
Indirect support for the hypothesis that xcex1-MSH is an endogenous regulator of basal skin tone comes from experiments in which removal of the pituitary gland induces lightening of the pigmentation of fish, amphibians, and lower mammals (Chavin, 1956, J. Exp. Zool. 133, 1-36; Smith, 1916, Science 44, 75-758; Allen, 1916, Science 44, 755-758; Rust, C. C., 1965, Gen. Comp. Endocrinol. 5, 222-231).
In addition, anecdotal clinical reports claim that humans become abnormally pale as a consequence of the loss of pituitary function due to disease or hypophysectomy (Felig et al., 1987, Endocrinology and Metabolism, McGraw-Hill Book Company, New York, 1-26). In humans, MSH is released not only from the pituitary, but also apparently by dermal keratinocytes following exposure to ultraviolet light (Luger, et al., 1993, Ann. N.Y. Acad. Sci. 680, 567-570). While it is well known that injection of MSH into animals or humans causes an increase in skin tone beyond basal levels, the contribution of endogenous xcex1-MSH to tonic baseline pigmentation has heretofore been unknown.
It has been shown that MSH effects an increase in tyrosinase activity of cultivated mouse melanoma cells. Synchronized cells were found to respond to MSH only in the G2 phase of the cell cycle. The binding of 1251-labeled MSH occurs predominantly in G2. Thus, it is proposed that MSH can activate melanoma cell adenylate cyclase by binding to an MSH receptor in the G2 phase (Varga et al., 1974, Proc. Natl. Acad. Sci. U.S.A., 71(5), 1590-3).
Thus, a practical antagonist of xcex1-MSH activity would provide a useful pharmacological agent for confirming the role of endogenous xcex1-MSH, and provide for methods of regulating all of the biological functions mediated by xcex1-MSH, including skin pigmentation tone and malignant melanoma cancer cells.
Recently, Jayawickreme et al., November 1994, J. of Biol. Chem. 269(47) 29846-29854, identified a series of polypeptide xcex1-MSH antagonists, e.g., octapeptides and nonapeptides. However, larger peptide molecules have limited ability to penetrate tissues of interest. Therefore, there remains a need for a small molecule xcex1-MSH antagonist, e.g., of less than 500 Da, better able to readily diffuse into tissue structures to regulate xcex1-MSH sensitive tissues.
The present invention provides peptide antagonists of xcex1-MSH and further provides methods for regulating the function of cells and tissues responsive to xcex1-MSH hormone. The invention provides for peptide antagonists according to the active xcex1-MSH peptides of table 1, infra. Thus, the invention provides for peptide antagonists of xcex1-MSH comprising an amino acid sequence of R-S-T, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, except that when S is Arg and T is Nle or an amide thereof, then R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; when S is Lys, D-Arg, Leu, Nle, Ala, Met, or Abu, and T is Nle or an amide thereof, then R is D-Trp; and when S is Arg, and T is Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or an amide thereof, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nle, Ala, Met, and Abu; and T is selected from the group consisting of Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg and amides thereof. The present invention also provides molecules having structures analogous to the disclosed peptide antagonists.
The invention also provides a method of inhibiting the activity of xcex1-MSH in an xcex1-MSH-responsive cell or tissue by contacting such a cell or tissue with a peptide antagonist of xcex1-MSH according to the invention.
The invention further provides a method of lightening the skin complexion of an animal by administering an effective amount of a peptide antagonist of xcex1-MSH according to the invention to an animal, e.g., a mammal such as a human, in need of such treatment. Methods of treatment are provided for pigmented spots, nevi, freckles, melasma and for local or systemic cosmetic lightening of the complexion. Administration may be topical or systemic.
The invention further provides a method of treating malignant melanoma by administering an effective amount of a peptide antagonist of xcex1-MSH according to the invention to an animal e.g., a mammal such as a human in need of such treatment.
The invention further provides a method of modulating the immune system by administering an effective amount of a peptide antagonist of xcex1-MSH according to the invention to an animal such as a human in need of such treatment.
In yet a further embodiment, the invention provides a pharmaceutical composition of a peptide antagonist according to the active xcex1-MSH antagonist peptides of table 1, infra. Thus, the invention provides antagonists of xcex1-MSH comprising an amino acid sequence of R-S-T, wherein R, S and T are amino acid residues and R is selected from the group consisting of D-Trp, D-Phe, D-Tyr, Ac-D-Trp, Trp and D-His, except that when S is Arg and T is Nle or an amide thereof, then R is D-Phe, D-Tyr, Ac-D-Trp, Trp or D-His; when S is Lys, D-Arg, Leu, Nle, Ala, Met, or Abu, and T is Nle or an amide thereof, then R is D-Trp; and when S is Arg, and T is Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg or D-Arg or an amide thereof, then R is D-Trp; S is selected from the group consisting of Arg, Lys, D-Arg, Leu, Nle, Ala, Met, and Abu; and T is selected from the group consisting of Leu, Nle, Nva, Met, D-Nle, Ile, Abu, Val, Arg, D-Arg or an amide thereof, together with a pharmaceutically acceptable carrier.
In an alternative embodiment, the invention provides a tripeptide antagonist of xcex1-melanocyte stimulating hormone that is identified by the method of preparing a combinatorial screening library comprising a library of tripeptide molecules of random structure. The random combinatorial screening library is then contacted with a test system for xcex1-melanocyte stimulating hormone activity, followed by identification of tripeptide molecules that antagonize the xcex1-melanocyte stimulating hormone activity.